Lack of selectivity of chemotherapeutic agents is a major problem in cancer treatment. Because highly toxic compounds are used in cancer chemotherapy, it is typically associated with severe side effects. Drug concentrations that would completely eradicate the tumor cannot be reached because of dose-limiting side effects such as gastrointestinal tract and bone marrow toxicity. In addition, tumors can develop resistance against anticancer agents after prolonged treatment. In modern drug development, targeting of cytotoxic drugs to the tumor site can be considered one of the primary goals.
One promising approach to obtain selectivity for tumor cells or tumor tissue is to exploit the existence of tumor-associated antigens, receptors, and other receptive moieties, which can serve as a target. Such a target may be upregulated or to some degree be specifically present in tumor tissue or in closely associated tissue, such as neovascular tissue, with respect to other tissues in order to achieve efficient targeting. Many targets have been identified and validated and several methods to identify and validate targets have been developed.1 
By coupling a ligand, e.g. an antibody or antibody fragment or a derivative thereof, for such a tumor-associated antigen, receptor, or other receptive moiety to a therapeutic or diagnostic agent, this agent can be selectively targeted to tumor tissue. In case the therapeutic or diagnostic moiety needs to be released at the tumor site, some kind of triggering mechanism may be present in the conjugate that is triggered when the conjugate has reached its target in order to release the payload. Such a triggering mechanism can for example be an enzymatic cleavage or a pH-dependent hydrolysis.2 Alternatively, release may occur non-specifically.
Another promising approach to obtain selectivity for tumor cells or tumor tissue is to exploit the existence of tumor-associated enzymes. A relatively high level of tumor-specific enzyme can convert a pharmacologically inactive prodrug, which consists of an enzymatic substrate directly or indirectly linked to the toxic drug, to the corresponding drug in the vicinity of or inside the tumor. Via this concept a high concentration of toxic anticancer agent can be selectively generated at the tumor site. All tumor cells may be killed if the dose is sufficiently high, which may decrease development of drug-resistant tumor cells.
There are several enzymes that are present at elevated levels in certain tumor tissues. One example is the enzyme β-glucuronidase, which is liberated from certain necrotic tumor areas. Furthermore, several proteolytic enzymes have been shown to be associated with tumor invasion and metastasis. Several proteases, like for example the cathepsins and proteases from the urokinase-type plasminogen activator (u-PA) system are all involved in tumor metastasis. The serine protease plasmin plays a key role in tumor invasion and metastasis. The proteolytically active form of plasmin is formed from its inactive pro-enzyme form plasminogen by u-PA. The tumor-associated presence of plasmin has been exploited for targeting of plasmin-cleavable conjugates or prodrugs.3 
Enzymes have also been transported to the vicinity of or inside target cells or target tissue via for example antibody-directed enzyme prodrug therapy (ADEPT)4, polymer-directed enzyme prodrug therapy (PDEPT) or macromolecular-directed enzyme prodrug therapy (MDEPT)5, virus-directed enzyme prodrug therapy (VDEPT)6, or gene-directed enzyme prodrug therapy (GDEPT)7.
Yet another promising approach to obtain selectivity for tumor cells or tumor tissue is to exploit the enhanced permeability and retention (EPR) effect. Through this EPR effect, macromolecules passively accumulate in solid tumors as a consequence of the disorganized pathology of angiogenic tumor vasculature with its discontinuous endothelium, leading to hyperpermeability to large macromolecules, and the lack of effective tumor lymphatic drainage.8 
By coupling therapeutic or diagnostic agents directly or indirectly to a macromolecule, e.g., a polymer such as for example poly[N-(2-hydroxypropyl)methacrylamide] (HPMA), poly-L-glutamic acid (PG), or polyethylene glycol (PEG), agents have been selectively targeted to tumor tissue. In case the therapeutic or diagnostic moiety needs to be released at the tumor site, some kind of triggering mechanism may be present in the conjugate that is triggered when the conjugate has reached its target in order to release the payload. Such a triggering mechanism can for example be an enzymatic cleavage or a pH-dependent hydrolysis.9 Alternatively, release may occur non-specifically.
Obviously, two or more targeting approaches such as the above-mentioned approaches to achieve tumor-selective delivery of the therapeutic or diagnostic agents can be combined into a single conjugate.
WO 02/083180 and WO 2004/043493 are relevant disclosures that describe targetable conjugates in which the use of a targeting moiety and the use of a specifier—a unit that can be conditionally cleaved or transformed—are combined to provide for optimal targeting of the one or more therapeutic or diagnostic moieties connected to the cleavable substrate via a self-eliminating spacer or spacer system.
The synthetic routes towards such conjugates comprise some disadvantages. The syntheses of these conjugates are composed of many synthetic steps. Furthermore, routes towards these conjugates regularly require the use of two or more orthogonal protecting groups that all need to be removed under mild conditions, as functional groups in the specifier, linker, and/or the therapeutic/diagnostic moiety or moieties require temporary protection and deprotection must be very mild to save structural integrity. Due to lack of suitable protecting groups, one may even not be able to synthesize some desired conjugates. In addition, new synthetic routes may need to be developed when new coupling strategies are required and the pool of protecting groups that can be chosen from is sometimes limited because of the functionalities and the reactive groups present in the compounds.
It can be understood that the synthetic routes towards conjugates that are structurally similar and that are used for purposes including, but not limited to, in vitro diagnostic assays, in vivo imaging, treatment or prevention of diseases, including cancer, improving the pharmacokinetic properties of agents, or in vivo/ex vivo controlled delivery of agents, may face the same or similar problems.
Thus there is a clear need in the art for improved conjugates that can be prepared with more ease (if they can be prepared at all according to other routes), in less synthetic steps, and according to more generally applicable routes in order to increase the yields and the scope of the conjugates and to reduce the amount of time required to prepare these conjugates.
The recitation of any reference in this section is not an admission that the reference is prior art to this application.